Reprogramming eukaryotic translation with ligand-responsive synthetic RNA switches
Open accessAbstract
Protein synthesis in eukaryotes is regulated by diverse reprogramming mechanisms that expand the coding capacity of individual genes. Here, we exploit one such mechanism, termed -1 programmed ribosomal frameshifting (-1 PRF), to engineer ligand-responsive RNA switches that regulate protein expression. First, efficient -1 PRF stimulatory RNA elements were discovered by in vitro selection; then, ligand-responsive switches were constructed by coupling -1 PRF stimulatory elements to RNA aptamers using rational design and directed evolution in Saccharomyces cerevisiae. We demonstrate that -1 PRF switches tightly control the relative stoichiometry of two distinct protein outputs from a single mRNA, exhibiting consistent ligand response across whole populations of cells. Furthermore, -1 PRF switches were applied to build single-mRNA logic gates and an apoptosis module in yeast. Together, these results showcase the potential for harnessing translation-reprogramming mechanisms for synthetic biology, and they establish -1 PRF switches as powerful RNA tools for controlling protein synthesis in eukaryotes.
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Two stabilizing RNA elements (Zika virus-derived xrRNA1 dumbbell and evopreQ1 pseudoknot) led to a robust increase in bcgRNA UMI counts, while protein knockdown was indistinguishable among six structures.
“While protein knockdown was indistinguishable for all six structures (Fig. 5b,c and Extended Data Fig. 9b), we found that two elements (Zika virus-derived xrRNA1 dumbbell33; evopreQ1 pseudoknot34) led to a robust increase in bcgRNA unique molecular identifier (UMI) counts (Fig. 5d).”
From Efficient combinatorial targeting of RNA transcripts in single cells with Cas13 RNA Perturb-seq (2023) · cites this paper as [34]
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- Authors
- Anzalone, A. V., Lin, A. J., Zairis, S., Rabadan, R., & Cornish, V. W.
- Affiliations
- Columbia University (US)
- Corresponding author
- Anzalone, Andrew V
- Cornish, Virginia W
- Venue
- Nature Methods
- Published
- 2016-03-21
- Volume
- 13
- Issue
- 5
- Pages
- 453-458
- ISSN
- 1548-7091 (ISSN-L)
- 1548-7105
- PMC
- PMC4850110
- OpenAlex
- W2302027613
- Cited by
- 33
- References
- 48
- Citations by year
- 2026: 2 · 2025: 3 · 2024: 1 · 2023: 2 · 2022: 4 · 2021: 4 · 2020: 1 · 2019: 6 · 2018: 2 · 2017: 3 · 2016: 5
- FWCI
- 2.77
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature Methods (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- Funders
- National Institutes of Health (TL1TR000082)
- National Cancer Institute (U54CA193313, F30CA174357)
- National Institute of General Medical Sciences (R01GM090126)
- National Institute of Allergy and Infectious Diseases (R01AI110794)
- National Center for Research Resources (S10RR027050)
- National Center for Advancing Translational Sciences
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Research and Splicing
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gene Regulatory Network Analysis
- Keywords
- RNA aptamers, in vitro selection, directed evolution, Saccharomyces cerevisiae, synthetic biology
- MeSH terms
- Biotechnology, Ligands, RNA, Fungal, RNA, Messenger, Saccharomyces cerevisiae, Protein Biosynthesis, Gene Expression Regulation, Fungal, Frameshifting, Ribosomal, Directed Molecular Evolution, Riboswitch, Cellular Reprogramming
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